Abstract The heart is the organ with the greatest fatty acid (FA) utilization and heart failure (HF) is almost always associated with alterations in cardiac lipid metabolism: diabetes and obesity increase FA use; failing hearts have reduced FA oxidation (FAO). FAs are needed in conditions of greater afterload, but in other situations excess lipid uptake leads to HF, a process sometimes referred to as lipotoxicity. This project focuses on how FAs are acquired by the heart and used for energy use, and why toxicity occurs when excess lipid accumulates. We have studied the roles of the triglyceride lipolysis enzyme lipoprotein lipase (LpL) and the FA transporter cluster of differentiation (CD) 36 in the movement of non-esterified FAs and lipoprotein-derived FAs into hearts by floxing the genes of each of these proteins. We found that endothelial CD36 is a major regulator of acute FA uptake by the heart. Although deletion of CD36 in either endothelial cells or cardiomyocytes leads to a marked reduction in lipid droplet (LD) accumulation during fasting, only the endothelial deletion led to an increase in mRNA levels of genes mediating insulin signaling and glucose uptake. Thus, CD36 actions differ in these two cell types. This revision has three aims that focuses on cardiac FA uptake and LD formation. In Aim 1, we will determine how changes in CD36 affect lipid uptake and LD formation. Aim 2 will compare the composition of LDs associated with toxicity and physiologic storage of triglyceride. Aim 3 will test whether HF due to adipose triglyceride lipase (ATGL) deficiency, a cause of human HF, can be corrected by CD36 deletion or inhibition. The overall objective of our studies is define the pathways required for FA uptake by the heart, to understand whether which forms of stored triglyceride are beneficial, and to define methods to treat lipotoxic heart disease.